Throughout this application, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
Immunochemical characterization of antibodies to alpha (1.fwdarw.6) dextran has given insights into the size and shape of the antibody combining site and the nature of the interaction between antibodies and antigen. In this regard, it would be useful to correlate the immunochemical properties of the anti-dextran antibodies with their primary structure. In the course of these studies, cDNAs from three monoclonal anti-alpha (1.fwdarw.6) dextran hybridoma cell lines, 14.6b.1, 5.54.4.24.1, and 19.22.1, were cloned (1) and the nucleotide sequences of the variable regions of the heavy chain (V.sub.H) and of the light chain (V.sub.L) determined (2) (see Table I on page 22 of this application). All synthesize an identical kappa light chain with the V.sub.kappa -OX1 germline gene (3) rearranged to the J.sub.kappa 2 segment and the heavy chains differ by only one or two amino acids in their complementarity-determining regions (CDRs). When compared to 14.6b.1, 5.54.4.24.1 and 19.22.1 have an identical Thr.fwdarw.Asn amino acid change at position 60 in the variable region of the heavy chain (V.sub.H); 5.54.4.24.1 has an additional change (Ser.fwdarw.Gly) at position 31 in complementarity-determining region 1 (CDR1). The changes in heavy chain sequence result in 5.54.4.24.1 and 19.22.1 having a (ten) 10 fold or greater reduction in their binding constant for both polymeric dextran and isomaltoheptaose (IM7) when compared to 14.6b.1 (Table I).
The Thr.fwdarw.Asn change in 5.54.4.24 and 19.22.1 leads to the loss of a potential N-linked glycosylation site (Asn 58-Tyr 59-Thr 60) present in 14.6b.1. One of the purposes of this study and the present invention was to determine whether this potential N-linked glycosylation site is glycosylated, and if so, whether the addition of carbohydrate to complementarity-determining region 2 (CDR2) affects the binding constant for dextran. It is difficult to demonstrate glycosylation of V.sub.H directly since both Immunoglobulin A (IgA) and Immunoglobulin M (IgM) isotypes are glycosylated within their C.sub.H 1 domains and carbohydrate present in Fd could be linked to either V.sub.H or constant region of the heavy chain (C.sub.H). Fd is the product resulting from the chemical or enzymatic cleavage of the antibody and comprises the heavy chain of the variable region and the heavy chain of the constant region of the antibody. Therefore, the three V.sub.H regions have been transferred to the human IgG.sub.4 constant region which is devoid of carbohydrate in its C.sub.H 1 domain. In this invention, the presence of carbohydrate is demonstrated to be within the V.sub.H of 14.6b.1. Comparison of the association constants for aglycosylated, tunicamycin treated and untreated antibodies shows that the presence of carbohydrate increases the apparent association constant (aKa) of 14.6b.1 for dextran. The effect on binding is unique to the carbohydrate present in V.sub.H since absence of carbohydrate from C.sub.H 2 does not change the aKa for dextran.
By introducing into an antibody a carbohydrate recognition site for the attachment of carbohydrate, purification of the antibody can be enhanced because the carbohydrate is attached to the outside of the antibody and thus, is more accessible to binding by lectin (purification).
In this invention, the carbohydrate content of an antibody may be modified by adding or deleting carbohydrate recognition sites in the constant region of the antibody. In so doing, effector functions of the antibody are modified. Carbohydrate recognition sites in the constant region can also serve as sites for labelling, e.g., radionuclides, such as .sup.125 I.